For breeding of animals, maximum fertilization is dependent upon precise insemination of sufficient numbers of healthy, viable sperm at the appropriate times. The decrease in fertilization due to poor timing of insemination may be a result of either a loss of sperm viability or a reduction in numbers of viable sperm available during the fertile life of the ovum. It is desirable to improve conception rates in artificially bred animals through the prolongation of viable sperm retention and release in the female reproductive tract.
Studies have already been undertaken by several laboratories to evaluate the efficiency of microencapsulation of spermatozoa as a means of enhancing sperm retention in the female following artificial insemination. Microencapsulation is based on the fact that large losses of sperm occur from and within the female during insemination, especially after the deposition of previously cryopreserved spermatozoa. The major causes of sperm disappearance appear to be leucocytic phagocytosis (engulfment of sperm by white blood cells which invade the uterus at the time of heat), and retrograde removal (expulsion of sperm from the uterus back through the cervix).
It has been postulated that microencapsulation will protect sperm from white blood cells and retrograde removal, and with appropriate capsule construction, could provide a prolonged release of sufficient numbers of sperm at a preprogrammed time to allow fertilization in a single insemination. Also, because sperm need not be cryopreserved prior to insemination, normal loss occurring as a consequence of thawing from the frozen state would not be a factor. This would allow for greater numbers of viable sperm to be deposited.
"Microencapsulation", as used herein, is defined as a process whereby small living cells are completely surrounded and enclosed by relatively inert materials to form a microcapsule. Microcapsules range in diameter from approximately 0.2 microns to several millimeters.
Microcapsules have been provided with both impermeable and semipermeable membranes, depending on the composition of the enclosed material and the use intended. The mechanism(s) for release were associated with leaching, erosion, rupture, or other such actions, depending upon construction of the capsule wall.
Nebel, et al., J. Anim. Sci., 60: 1631 (June 1985) reported the microencapsulation of bovine spermatozoa. Their microcapsules differed from those of the present invention in that they were not designed to assume a liquid state at a particular temperature. Sperm cells were suspended in a sodium alignate and fine droplets of this suspension were produced using a syringe pump extrusion technique. The droplets were collected in a calcium chloride solution which results in an immediate gellation of the droplets, thus producing a shape-retaining high viscosity mold for the microcapsules. A semi-permeable membrane was applied to the droplets by suspending them in a solution of polylysine, thus forming the microcapsules. The gelled suspension of sperm inside the capsules was then liquified by exposing the capsules to a solution of sodium citrate, rather than by raising the temperature to body temperature. Once a sperm suspension in the capsule was liquified, the sperm resumed the mobility which had been arrested temporarily by gellation.
However, the sperm showed diminished motile life following such encapsulation and total loss of fertilizability. Further studies indicated that when biodegradable microcapsules were used, the retention of the sperm was poor. The biodegradable microcapsules were also prone to early rupture and retrograde removal.
Drug delivery means which are solid or gel-like at room temperature and liquid at body temperature are known. These do not allow for providing nutrients and oxygen to living cells to maintain their viability. Also, they do not provide or require a sharp phase transition with respect to temperature.
The present invention contemplates the encapsulation of living, viable sperm such that the sperm's motility, viability, retention and release in the female reproductive tract is not significantly impaired.